Summary/Abstract Our preliminary data suggest the existence of as-of-yet unidentified regulator of the important inflammatory pathway controlled by Toll-like Receptor 4 (TLR4), and the central goals of this project are to identify and validate candidate regulators using comprehensive genome-scale analyses. TLR4 induces potent inflammation in response to lipid A, the core component of bacterial endotoxin. While this response provides a first line of defense against bacterial infection, excessive TLR4 signaling promotes disease. Thus, understanding how TLR4 signaling is regulated is of central importance for understanding both health and disease processes. Despite the substantial progress made in elucidating many aspects of TLR4 biology, the mechanisms by which signaling is suppressed in the absence of ligand and then initiated upon the formation of a signaling complex remains a relatively less understood aspect of TLR4's mechanism. In recent preliminary work, we inferred the existence of a currently unidentified negative regulator of TLR4 signaling, which we term the TLR4 inhibitory complex (TIC) and which operates via a mechanism distinct from that of any known negative regulator of TLR4 signaling. We hypothesize that TIC binds TLR4, preventing it from signaling in an otherwise constitutive fashion, such that deficiencies or disruptions of TIC components may drive chronic TLR4 signaling. Indeed, this switch-like TLR4 signaling hypothesis is consistent with the peculiar observation that distinct non-canonical ligands, such as metals, can also trigger TLR4 signaling. Understanding how such negative regulation is disrupted, for example in the context of genetic polymorphisms, could also shed important diagnostic light on many diseases involving chronic inflammation. To investigate this TIC hypothesis, in this project we propose to both identify putative components of TIC and validate their involvement in TLR4-driven inflammation. In our first aim, we will identify candidate TIC components, which negatively regulate constitutive TLR4 signaling, via an unbiased genome-scale knock-out screen. We will harness the power of CRISPR/Cas9-based functional genomic screens to investigate the knockout of 19,000 protein-coding genes, evaluate the impacts of these knockouts on TLR4 signaling, and generate a ranked list of putative TIC components. In our second aim, we will validate and evaluate the role of candidate TIC components in TLR4 signaling. We will investigate whether depletion of putative TIC components identified in Aim 1 confers constitutive TLR4 signaling. Validated targets will be further investigated using biochemical assays to investigate whether TIC components physically associate with TLR4 in both the presence and absence of endotoxin, which would ultimately confirm a role for putative TIC components in ligand-dependent TLR4 signaling. If successful, this project would contribute novel understanding of an important driver of inflammation and could identify novel target(s) of value for diagnosing and treating infections and inflammatory diseases.